No 14. plant physiology and fruit secondary metabolites of canistel (pouteria campechiana).

World Applied Sciences Journal 33 (12): 1908-1914, 2015
ISSN 1818-4952
© IDOSI Publications, 2015
DOI: 10.5829/idosi.wasj.2015.33.12.15625
Corresponding Author: H. Mehraj, The United Graduate School of Agricultural Sciences,
Ehime University, Ehime 790-8556, Japan.
1908
Plant Physiology and Fruit Secondary Metabolites of
Canistel (Pouteria campechiana)
H. Mehraj, R.K. Sikder, U. Mayda, T. Taufique and A.F.M. Jamal Uddin1 2 3 4 4
The United Graduate School of Agricultural Sciences, Ehime University, Ehime 790-8556, Japan1
Horticulture Development Division, BADC, Dhaka-1000, Bangladesh2
Department of Botany, Jahangirnagar University, Savar, Dhaka, Bangladesh3
Deaprtment of Horticulture, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh4
Abstract: Canistel (Pouteria campechiana) is a newly cultivated fruits in Bangladesh. From this study, we tried
to find out plant physiology and also secondary metabolites in hydro-alcoholic, methanol and aqueous extract
of canistel fruit. Plant physiological characters were determined by respective instruments and fruit
phytochemicals were screened using standard methods. SPAD reading (47.9%), photosynthetic rate (9.6
µmolm s ), stomatal conductance of H O (0.8 µmolm s ), CO references (348.4 vpm), H O references as2 1 2 1
2 2 2
partial pressure (33.8 mBar) and P.A.R incident on leaf surface (271.1 µmolm s ) was found out. It yielded2 1
123.6 kg fruit/plant while fruit had 11.6% degree of brix and 61.3 mg/100 g fruit Vit.-C with 13.3% moisture.
Alkaloids, glycosides, carbohydrates, tannins, terpenoids, steroids, reducing sugar, phlobatannins, proteins,
amino acids, lipids, fats and acidic compounds were found to be present but flavonoids, phenolics and
saponins were found absent on canistel fruit. Canistel fruit have a number of secondary metabolites and
identification of these phytochemicals may be helpful for phytochemists and pharmacologists in future.
Key words: Fruit Alcoholic Aqueous Extract Phytochemicals
INTRODUCTION MATERIALS AND METHODS
Canistel (Pouteria campechiana) is a new fruit for Experiment was conducted at Horticulture
Bangladesh also known as yellow sapote or egg-fruit Development Centre, Bangladesh Agricultural
native to Central America but distributed to tropical and Development Corporation (BADC), Tangail and
subtropical areas [1] that belongs to Sapotaceae family. 2abiotech, Department of Horticulture, Sher-e-Bangla
Dietary phytochemicals are found in different parts of the Agricultural University, Dhaka-1207, Bangladesh during
plants like fruits, vegetables, legumes, grains, nuts, seeds, May 2012 to 2013.
fungi, herbs and spices [2, 3]. Any fruits have the
potentiality of excellent medicinal plant metabolites like Physiological, Physical and Chemical Properties of
alkaloids, glycosides, terpenoids, tannins, phenolics,
flavonoids, steroids, saponins and lignans [4]. Canistel
fruit may also contain essential secondary metabolites.
Generally, phytochemical surveys are done to identify the
chemical, toxicological and pharmacological aspects of a
plant or plant parts. Canistel plant physiology and fruit
secondary metabolites are till now unknown. So, we
conducted this study mainly to find out the plant
physiology and fruit secondary metabolites of canistel
fruits.
Plant: Leaf area and chlorophyll was measured by CL-202
Leaf Area Meter and SPAD 502. Infrared gas analyzer
(IRGA)-LC pro+ Photosynthesis System was used to
determine photosynthetic rate, stomatal conductance of
H O, CO references, H O references as partial pressure,2 2 2
P.A.R incident on leaf surface. Brix percentages were
measured by Portable Refractometer (ERMA, Tokyo,
Japan). Vit-C content was estimated by Oxidation
Reduction Titration Method. 10 g sample was taken and
dried in air oven at 105°C until to gain a constant weight

World Appl. Sci. J., 33 (12): 1908-1914, 2015
1909
and dried sample weighted again. This procedure was yellow precipitate (with Hager’s reagent i.e., saturated
repeated 5 times for accuracy. Moisture content (%) was picric acid) was regarded as positive for the presence of
estimated by following formula: alkaloids.
Moisture (%) = {(W -W ) ÷ (W -W )} × 100 Glycosides: By (a) FeCl , (b) Keller killiani’s and (c)1 2 1 0
Where, W = weight of empty petri dish (g); W = weight0 1
of petri dish with sample before drying (g) and W = (a) FeCl solution were added on extracts and dipped2
weight of petri dish with sample after drying (g). in boiling water for 5 minutes then cooled and shaken
Secondary Metabolites Determination: Canistel fruits layer was separated and ammonia solution was
were washed with water and cut into small pieces, dried added. Rose-pink colour in ammoniacal layer
and powdered. Extraction was performed by cold confirmed the presence of glycosides. (b) 2 ml of
maceration method. glacial acetic acid with FeCl (1 drop) was added to 5
Hydro-alcoholic Extract and Aqueous Extract Brown ring denoted deoxysugar characteristic of
Preparation: About 1000 g of fruit powder were immersed cardenolides and violet ring may appear below the
in hydro-alcoholic (80% ethanol) solution in a 5000 ml flat brown ring. In acetic acid layer, greenish-blue ring
bottom flask and cold extracted for 7 days with occasional may form throughout thin layer confirmed the
shaking and warming. After that, clear filtrate was presence of cardiac glycosides [9]. (c) Benzene and
obtained by Buchner funnel filtering. Filtrate was further 10% ammonia (10 ml of each) was added with 3 ml of
concentrated by vacuum distillation, cooled, transferred extract. Pink, red or violet color in lower phase of
into petri dish and dried in an oven at 60°C for five ammonia assured the presence of anthraquinone i.e.,
minutes. To remove excessive moisture hydro-alcoholic glycosides.
extract was kept in a desiccator for 15 days [5]. Similarly
aqueous extract was prepared only exception that fruit Carbohydrates: By (a) Molisch and (b) Fehling's test.
powders were immersed on aqueous solution.
Methanol Extraction: 10 g of fruit powder was dissolved distilled water and filtered individually. After treating
in 100 ml of methanol and shake them on a rotary shaker the filtrates with 2 drops of alcoholic á-naphthol
at 190-220 rpm for 24 h. Supernatant was collected slowly solution and H SO (2 ml conc.) violet ring was
and stored at 4°C in airtight bottles. formed at junction which pointed out the presence of
Qualitative Phytochemical Analysis: Phytochemicals hydrolyzing with diluted HCl, neutralizing with alkali
were identified by testing hydro-alcoholic, methanol and and heating with Fehlings A and B solutions,
aqueous extracts [6, 7, 8] as following the test included formation of red precipitation indicated the presence
below. of carbohydrates (Fehling's test).
Alkaloids: By (a) Dragendroff's, (b) Wagner's, (c) Mayer's Flavonoids: By (a) Lead acetate (b) Alkali (c) Conc. H SO
treatment and (d) FeCl
ml acid alcohol, boiled and filtered. Filtrate (5 ml) was
added to dilute ammonia (2 ml) and chloroform (5 ml) then (a) and (b) Few drops lead acetate (for lead acetate
shaken gently to extract alkaloidal base. Chloroform layer test) and NaOH (alkali test) solution was put in
was extracted with 10 ml of acetic acid. This was divided extract solution then yellow color precipitate
into four portions and Dragendroff's test, Wagner's test, formation directly indicated the presence of
Mayer's test and Hager's reagents were added to each flavonoids (by lead acetate test) but if yellow color
portion. The formation of cream (with Mayer’s reagent i.e., becomes colorless by the addition of dilute acid
Potassium Mercuric iodide), brown/reddish brown (with confirmed the presence of flavonoids (by alkali test).
Wagner’s reagent i.e., Iodine potassium iodide and with (c) Conc. H SO (1 ml) and dilute ammonia (5 ml) was
Draggendorff’s reagent i.e. potassium bismuth iodide) and added on the extract and appearances of the yellow
3
Borntrager’s test (Anthraquinones)
3
with equal volume of benzene. After that benzene
3
ml extract then underlayed with 1 ml of H SO (conc.).2 4
(a) 0.5 mg of each extracts were dissolved in 5 ml
2 4
carbohydrates (Molisch’s Test). (b) Filtrates
2 4
and (d) Hager's test (a-d) 0.5 g of extract was diluted to 10 3.
2 4

World Appl. Sci. J., 33 (12): 1908-1914, 2015
1910
color that disappear on standing was the indicator for Protein and Amino Acid: By (a) Xanthoproteic, (b) Biuret
presence of flavonoids. (d) Few drops of FeCl were3
added on extract then formation of blackish red color
indicating the presence of flavonoids.
Tannins and Phenolics: By (a) FeCl and (b) gelatin test.3
(a) Addition of 2-3 drops FeCl (2%) reagent to 1ml3
of extract for formation of dark blue or greenish black
color which indicated presence of tannins. (b) 2-3
drops gelatin (10%) were added to 1ml extract and
white precipitation assured the presence of phenolic
compounds.
Terpinoids: By (a) terpine and (b) triterpine test.
(a) Test for terpenoids was done by dissolving two
or three granules of tin metal in 2 ml thionyl chloride
solution and then, add 1 ml of the extract into the test
tube. The formation of a pink colour indicates the
presence of terpenoids. (b) Extracts were treated with
chloroform and filtered. The filtrates were treated with
a few drops of H SO (conc.), shaken and allowed to2 4
stand. Golden yellow colour indicated the presence
of triterpines.
Steroids: By Salkowski test. About 5 mg extract was
dissolved in 2 ml chloroform then 2 ml H SO (conc.) was2 4
added while red upper layer and yellow lower layer with
green fluorescence, indicated the presence of steroids.
Saponins: By foam test. About 1 ml of extract was taken
which was diluted with 20 ml distilled water and then
shaken in a graduated cylinder for 15 minutes. 1.0 cm layer
of foam indicates the presence of saponins [7].
Reducing Sugar: By (a) Fehling test and
(b) Molisch’s test.
(a) 0.50 g of fruit was added in 5 ml of distilled water
and 1 ml of ethanol was mixed. Fehling solution (1 ml
of each Fehling solution A and B) was heated to boil
and poured it into aqueous ethanol extract. Color
reaction indicated the positive result. (b) Few drops
of Molisch’s reagent were added with dilute extracts
and heated for 30 minutes. Brick red precipitate
confirmed the presence of reducing sugar.
Phlobatannins: Fruit powder solution was filtered 1%
aqueous HCl was added and then boiled by Hot plate
stirrer. Red colored precipitate confirmed a positive result.
and (c) Nin hydrin test
(a) Few drops of HNO (conc.) were added to the3
extracts. Yellow color observation indicated the
presence of protein. (b) 1 ml NaOH (10%) were added
to the extract and heated. After that a drop of CuSO4
(0.7%) was added and formation of purplish violet
colour confirmed proteins. (c) Distilled water (1 ml)
and Nin hydrin (300 ìl) was added with extract (300 ìl)
then boiled for 5-10 minutes. Dark purple color
indicated the presence of amino acids.
Lipid and Fat Test: A small quantity of powdered drug
was rubbed on a clean and neat filter paper and observed
for a permanent translucent strain.
Acidic Compounds: A bit of NaHCO was added with 3003
ìl of extract. Gas bubbles ensured the presence of acidic
compounds.
Each test was done three times and positive results
from each tests was marked by positive sign while
negative result was marked by negative sign.
RESULTS AND DISCUSSION
Canistel plant had 179.3 cm leaf area and 47.9%2
leaves SPAD reading while A (9.6 µmolm s ), g (0.82 1
s
µmolm s ), C (348.4 vpm), e (33.8 mBar) Q (271.12 1
ref ref leaf
µmolm s ) was also found (Table 1). Similar2 1
physiological characteristics were determined in
bougainvillea [10] and chilli [11]. Low chlorophyll content
in leaves caused higher yields also reduce heat load at top
canopy and may increase available nutrients for plant [12].
Stomatal conductance is important for CO gas exchanges2
and outflow of water in vapor form because it also
restricts the intake of CO [13]. More leaf photosynthesis2
increases biomass productivity [14] and yield [15] but has
co-relation on plant development phase [16, 17].
Canistel fruit was 55.1 mm in length, 29.9 mm in
diameter and 26.8 g of a single fruit weight whereas
yielded 123.6 kg/plant. Fruits of canistel had 11.6% brix,
61.3 mg/100 g fruit Vit.-C. Moisture of the canistel fruit
was 13.3% (Table 1).
Canistel fruit’s were focused to qualitative
phytochemical screening for secondary metabolites like
alkaloids, glycosides, carbohydrates, flavonoids, tannins
and phenolics, terpinoids, steroids, saponins, reducing
sugar, phlobatannins, protein, acidic compounds, lipid
and fat (Table 2). In the current study, hydro-alcoholic,

World Appl. Sci. J., 33 (12): 1908-1914, 2015
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Table 1: Some physiological, physical and chemical properties of canistel
(Pouteria campechiana) fruits
Parameters characteristics Quantity
Physiological properties of plant
Leaf area 179.3 cm2
Chlorophyll content 47.9%
Photosynthetic rate (A) 9.6 µmolm s2 1
Stomatal conductance of H O (gs) 0.8 µmolm s2
2 1
CO references (Cref) 348.4 vpm2
H O references as partial pressure (e ) 33.8 mBar2 ref
P.A.R incident on leaf surface (Q ) 271.1µmolm sleaf
2 1
Physical and chemical properties of fruit
Fruit length 55.1 mm
Fruit diameter 29.9 mm
Single fruit weight 26.8 g
Yield/plant 123.6 kg
Brix 11.6%
Moisture 13.3%
Vit-C 61.3mg/100g fruit
Table 2: Presence or absence of secondary metabolites of canistel fruits
Test results for hydro-alcoholic,
Name of the tests methanol and aqueous extract Remarks
Alkaloid
Mayer’s reagent ++ Alkaloid present
Wagner’s reagent ++
Dragendroff’s reagent ++
Hager's test ++
Glycoside
FeCl test ++ Glycoside present3
Keller killianis test ++
Borntrager’s test --
Carbohydrate
Molisch’s test ++ Carbohydrate present
Felhing’s test ++
Flavonoid
Lead acetate test -- Flavonoid absent
Alkali test ++
Conc. H SO test --2 4
FeCl test --3
Tannins and phenolics
FeCl test ++ Tannic present but3
phenolics absent
Gelatin test --
Terpenoid
Test for Terpine ++ Terpenoid present
Test for Triterpine ++
Steroid
alkowski’s test ++ Steroid present
Saponin
Foam Test -- Saponin absent
Reducing sugar
Fehling test ++ Reducing sugar present
Molisch’s test ++
Phlobatannins ++ Phlobatannins present
Protein and amino acid
Xanthoproteic test ++ Protein present
Biuret test ++
Nin hydrin test ++ Amino acid present
Lipid and Fat ++ Lipid and fat present
Acidic compounds ++ Acidic compounds
present
++ = present and -- = absent
methanol and aqueous extracts showed similar results
for canistel fruit. Secondary metabolites are
pharmacologically active [18] and also very important for
disease prevention and health promotion. Similarly
phytochemical analysis was done in Asparagus spears
[19], citrus [20], Phaleria macrocarpa [21], medicinal
plants [22], Acalypha ornate [23], Albizzia lebbeck [24],
Ocimum americanum [25], Nerium oleander and
Momordica charantia [26], Pyrus pashim [27], Jatropha
[28] and various plant parts of Quetta Balochistan [29,
30]. They all mentioned the presence/absence of alkaloids,
glycosides, carbohydrates, flavonoids, tannins, terpenes,
phenolics, steroids, saponins, reducing sugar,
phlobatannins, protein, amino acid, lipid and fat also
amino acid. Alkaloid has pharmacological activities [31]
because it poses anti-bacterial and analgesic properties
[32] also used for creation of potent pain killer [33].
Glycosides are cardio active drugs [34] because it inhibits
Na and K pump by rising availability of Na and Ca to+ + + +
heart muscles which gets better cardiac output and lessen
heart expansion [35]. Presence of alkaloids and glycoside
on canistel fruit (Table 2) is very important and dietary
intake may prevent heart failure and cardiac arrhythmia.
Flavanoids and tannins are used for the treatment of
intestinal disorders also have anti-microbial, anti-
inflammatory, anti-allergic, anti-cancer, anti-neoplastic
activities [36]. Tannins also act as primary antioxidants or
free radical scavengers that may make the canistel
antioxidant capacity and is able to inhibit HIV [37]. Dietary
flavonoids also inhibit heart diseases, inflammation and
tumor growth but phytochemical analysis of the canistel
fruit did not show the positive result for flavonoids while
tannins were found to be present on canistel fruit (Table
2). Terpinoids are natural lipids and many of them are
commercially important due to their flavors and
fragrances; also important for agricultural produces that
ensure flavor [38]. It acted as a phytoalexins in plant
defense against natural enemies [39] also have medicinal
properties like anti-carcinogenic, anti-ulcer, hepaticidal,
anti-microbial or diuretic, anti-malarial drug and anti-
cancer drug [40] while terpenes may act as
phytohormones of plants also slower the cancer cell
growth. Presence of terpenoid in canistel fruit (Table 2)
indicated that consumption of this fruit may protect the
Bangladeshi people from some risky diseases. Dietary
intakes of steroid can increases anti-microbial [36], anti-
bacterial [41, 42] and anti-viral [43] properties in human
body and that were present in canistel fruit (Table 2).
Saponins played important role for hypercholesterolemia,

World Appl. Sci. J., 33 (12): 1908-1914, 2015
1912
hyperglycemia, as antioxidants, anti-cancers, anti-fungal, 5. Mandal, S.C., S. Lakshmi and T. Murugesan, 2000.
anti-bacterial, anti-inflammatory and in weight loss
treatments [44] that was not found in canistel fruit
(Table 2). Phlobatannins are present in canistel fruits and
it may act as anti-inflammatory and analgesic [45] and
antioxidant [46]. Presence of carbohydrates, reducing
sugar proteins, amino acid, lipid and fat makes canistel
fruit as a good food source. This study made conformity
that dietary intake of canistel fruit may help to improve
health of Bangladeshi people and will help to promote the
herbal drug not only in Bangladesh but also to the whole
world.
CONCLUSIONS
Physiological and phytochemical assessment gives
useful information about canistel fruits. Pouteria
campechiana was found to be rich in secondary
metabolites and may have used for treating several
diseases. All tested phytochemicals except flavonoids,
phenolics and saponins were present in canistel fruits that
have curative properties. We suggest to Bangladeshi
peoples for dietary intake of canistel fruit that will help to
prevent them from many diseases. However, further study
could be conducted to quantify the secondary
metabolites also isolation for pharmaceutical uses.
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